CN102000028B - Solamargine liposome, preparation method and usage thereof - Google Patents
Solamargine liposome, preparation method and usage thereof Download PDFInfo
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Abstract
The invention relates to solamargine liposome, which is characterized in that the liposome comprises the following raw materials: 1 part by weight of solamargine, 8 to 80 parts by weight of phospholipid, 2 to 20 parts by weight of cholesterol and 1 to 10 parts by weight of emulsifier. The invention also discloses a preparation method of the solamargine liposome. The invention has the advantages that the solamargine is coated between the two phospholipid layers of the liposome so that the water solubility is enhanced; the physical stability of the solamargine liposome is good; and the solamargine liposome can be stored for a longer time. The preparation method of the solamargine liposome is simple and mature, and is convenient to industrial production.
Description
Technical field
The present invention relates to a kind of alkaloid liposome, particularly a kind of solamargine liposome; The invention still further relates to the preparation method and its usage of this solamargine liposome.
Background technology
Solamargine, English name: solamargine, molecular formula: C
45H
73NO
15, structural formula:
Solamargine is a white powder; Water insoluble; Be soluble in methanol; Can extract refining forming from dry aerial parts disclosed any extraction process through prior art of plant of Solanaceae Herba Solani Nigri Solanum nigrum L., also can be buied by market, the content of solamargine preferably reaches more than 95%.The content of solamargine in herb of Herba Solani Nigri is at most with the immature fruit, can reach 4.2%.
Herba Solani Nigri is the dry aerial parts of plant of Solanaceae Herba Solani Nigri Solanum nigrum L., and bitter in the mouth, little sweet, cold in nature is returned lung, liver, stomach warp; The function heat-clearing and toxic substances removing, promoting blood circulation and detumescence.The record of many classics of TCM, the length of the existing detoxifcation inducing diuresis to remove edema of Herba Solani Nigri has the merit of anticancer blood stasis-eliminating and stagnation-dissipating again, has the wonderful of strengthening vital QI to eliminate pathogenic factors concurrently, and killing three birds with one stone is so be usually used in treatment for cancer on the tcm clinical practice.Discover that glycoalkaloids such as solasonine, solamargine are the main anticancer active constituent of Herba Solani Nigri, (peace is of heap of stone, Tang Jingtian; Liu new people, etc. Herba Solani Nigri Anticancer Effect and Mechanism progress [J], CHINA JOURNAL OF CHINESE MATERIA MEDICA; 2006; 31 (5): 1225-1226.), research thinks that the antitumor mechanism of Herba Solani Nigri mainly is that alkaloid in the Herba Solani Nigri can be through changing the 26S Proteasome Structure and Function of cell membrane, influence DNA and RNA synthesize and the change cell cycle distribution suppresses tumor.But solamargine is water insoluble, and certain toxic and side effects such as haemolysis are arranged, and its application as monomer medicine has been produced obstacle.
Liposome is meant that by phospholipid be the vesicle that film material and additives are formed; Be a kind of bilayer folliculus of similar biofilm structure; Can seal water solublity or fat-soluble medicine, its film material is biodegradation and avirulence and immunogenicity in vivo, thereby can be used as pharmaceutical carrier.Therefore liposome has the raising stability of drug, reduces drug dose, reduces the effect of drug toxicity, can also slow down drug release simultaneously, lowers elimination speed in the body.Because liposome is a kind of exogenous material; Get into the picked-up that can receive system of defense in the body in the body, thereby in the normal human, liposome mainly can be in position enrichments such as liver, spleen, lungs; Appearance can also be played the effect of passive target carrier than higher local drug concentration.As the carrier of antitumor drug, it can reduce the distribution of medicine in normal structure, increases the accumulation of medicine at tumor tissues, thereby improves the therapeutic index of medicine.In view of the special performance of liposome, its research is just being located the flourish stage at present, the liposome of some medicines has been used for multiple route of administration and preparation, and has carried out clinical practice.Present no-trump solamargine is still processed the relevant report of liposome.
Summary of the invention
Technical problem to be solved by this invention is the deficiency to prior art, provides a kind of physical stability good, can improve the water solublity of solamargine, reduces the solamargine liposome of its toxic and side effects.
Another technical problem to be solved by this invention has provided the method for preparing of above-mentioned solamargine liposome.
Another technical problem to be solved by this invention has provided the purposes of above-mentioned solamargine liposome.
Technical problem to be solved by this invention is to realize through following technical scheme.The present invention is a kind of solamargine liposome, is characterized in: it is made up of the following weight proportion raw material,
Solamargine 1; Phospholipid 8 ~ 80;
In the above-described solamargine liposome technology scheme, optimized technical scheme is:
1, the preferred weight proportion of each raw material is:
Solamargine 1; Phosphatidase 11 5 ~ 25;
2, solamargine of the present invention can be that monomer can be an extract also, and the content of solamargine is preferably greater than 80% in the extract, and most preferred content is greater than 95% solamargine; Described solamargine can be solamargine monomer or the extract that from the Chinese crude drug Herba Solani Nigri, extracts, commercially available solamargine monomer or the extract perhaps directly bought from market.
3, described phospholipid can be natural extract phospholipid, semi-synthetic or complete synthesis phospholipid, and is preferred: soybean lecithin, Ovum Gallus domesticus Flavus lecithin, hydrogenated soy phosphatidyl choline, hydrogenated yolk lecithin, two Semen Myristicae phosphatidylcholine, two Semen Myristicae phosphatidyl glycerol, dioleoyl phospholipid phatidylcholine, two palmityl phosphatidic acid, two palmityl phosphatidyl cholines or two palmityl phosphatidyl glycerols.Phospholipid can be selected from the commodity of listing, also can make by the prior art disclosed method.
4, the preferred tween of described emulsifying agent, poloxamer, Polyethylene Glycol or N, the mixture that one or more in the dinethylformamide are formed by proper ratio.
Technical problem to be solved by this invention can also further realize through following technical scheme.
Solamargine liposome of the present invention can prepare through following method:
Method one:
(1) use water for injection secure ph is 3 ~ 6.5 faintly acid buffer salt solution, and solamargine, emulsifying agent are dissolved in this buffer, gets water;
(2) take by weighing phospholipid, cholesterol in said ratio, be dissolved in the organic solvent, get organic facies;
(3) with water with after organic facies is mixed, adopt reverse evaporation or film dispersion method or injection method to handle earlier, ultrasonicly then make its dispersion, promptly get the solamargine liposome.
Method two:
(1) use water for injection secure ph is 3 ~ 6.5 faintly acid buffer salt solution, and emulsifying agent is dissolved in this buffer, gets water;
(2) take by weighing phospholipid, cholesterol, solamargine in said ratio, be dissolved in the organic solvent, get organic facies;
(3) with water with after organic facies is mixed, adopt reverse evaporation or film dispersion method or injection method to handle earlier, ultrasonicly then make its dispersion, promptly get the solamargine liposome.
In the method for preparing one and method two technical scheme of above-described solamargine liposome: described faintly acid buffer salt solution is PBS, citrate buffer solution; As: sodium hydrogen phosphate-potassium phosphate buffer, sodium hydrogen phosphate-citrate buffer solution or citric acid-sodium citrate buffer, pH value are 3 ~ 6.5.The mixed liquor of one or more compositions in the preferred chloroform of described organic solvent, ether, dichloromethane, normal hexane, ethanol, the methanol.
Solamargine liposome of the present invention can be applied to prepare in the medicine of treating hepatocarcinoma, pulmonary carcinoma and other cancer.
Solamargine liposome and method for making thereof that the present invention makes have the following advantages:
1, this method for preparing is simple, and phospholipid has excellent biological compatibility, and is biodegradable, and the source is wide, and cost is low, and liposome does not have immunosuppressive action to human body, and toxicity is less.
2, solamargine is wrapped between the phospholipid bilayer of liposome, has improved its water solublity.
3, the physical stability of solamargine liposome is good, can place the long period.
4, this solamargine method for preparing lipidosome is simple, and maturation is convenient to suitability for industrialized production.
5, solamargine is made lipidosome injection; Compare with common solamargine injection; Solamargine liposome haemolysis, muscle and vascular stimulation all improve, and can improve zooperal antitumous effect to a certain extent through targeting.
The present invention has carried out following further investigation to the pharmacological action of solamargine liposome.
Experiment 1.The hemolytic experiment of solamargine liposome.
1 experiment material.
1.1 medicine.
The solamargine injection, concentration 0.76mg/ml; Solamargine liposome A (according to embodiment 1 preparation), 1.0mg/ml; Solamargine liposome B (according to embodiment 3 preparations), 1.0mg/ml.
1.2 animal.1 of livid purple blue rabbit, male and female are regardless of, body weight 1.8 ~ 2.2kg.
2 experimental techniques.
2.1 medicine preparation.
Get solamargine injection and solamargine liposome A, B is diluted to three concentration: 0.05mg/ml, 0.1mg/ml, 0.15mg/ml with normal saline respectively, and is subsequent use as test sample, each sample adds to be done one and repeats pipe and do haemolysis inspection.
2.2 experimental technique.
Get 1 of livid purple blue rabbit, the about 10ml of heart extracting blood adds bead and stirs 10min removal Fibrinogen in conical flask, makes it to become defibrinated blood.Defibrinated blood is moved in the teat glass, and every test tube adds the about 5ml of normal saline, and (2000 commentaries on classics/min), remove supernatant add normal saline 5ml to centrifugal 5min behind the mixing again, and mixing is centrifugal.2～3 times so repeatedly, be water white transparency (do not show red) to supernatant till.The erythrocyte of gained by volume is mixed with 2% red blood cell suspension with normal saline, supplies experiment to use.
Get clean tube, be arranged on the test tube rack after numbering respectively.Press the various test solutions of adding shown in Figure 1, wherein manage negative control tube No. 1, manage positive control tube No. 2, each sample repeats a pipe.Behind the various test solution mixings, place 37 ℃ ± 0.5 ℃ calorstat to carry out incubation immediately, beginning was whenever observed 1 time at a distance from 15 minutes, and continuous 3 hours, per 2～3 h observation once after 3 hours.After 12 hours, get the supernatant (about 2.5ml) of respectively managing solution, 1000r after 10min is centrifugal, draws supernatant, on spectrophotometer, reads and respectively manages the OD value, measures wavelength 545nm, returns to zero with distilled water.
2.3 the result observes and calculates.
If the solution in the test is clear and bright redness, the pipe end, is acellular residual or have a small amount of erythrocyte residual, and showing has haemolysis to take place; All sink like erythrocyte, supernatant liquid achromatism and clarity shows that no haemolysis takes place.
If in the solution brownish red or rufous flocculent deposit are arranged, do not disperse after the jolting, showing has red blood cell condensation to take place.If any the phenomenon of red blood cell condensation, can further judge it is true cohesion or pseudo agglutination by purgation.If condensation product again can homodisperse after test tube vibration; Or condensation product is placed on the microscope slide; Drip 2 0.9% sodium chloride solutions at the coverslip edge; Put microscopically and observe, the cohesion erythrocyte can be pseudo agglutination by the person of breaking up, if condensation product is not shaken diffusing or on slide, be not true cohesion by the person of breaking up.
Do not take place with cohesion when the negative control pipe has haemolysis, when the positive control pipe has haemolysis to take place,, then tried thing and can inject use if haemolysis and cohesion did not take place in 3 hours the solution that is tried in the property management; If tried solution in the property management in 3 hours, takes place haemolysis with (or) condense, then tried thing and should not be injected use.
2.4 spectrophotography.
Hemolysis rate (%) with each developmental tube of computes:
Hemolysis rate (%)=(OD
t-OD
NC)/(OD
PC-OD
NC) * 100%
OD wherein
t: the developmental tube absorbance; OD
NC: negative control pipe absorbance; OD
PC: positive control pipe absorbance.
Show have haemolysis to take place with reference to evaluation criterion: hemolysis rate ﹥ 5%, and carry out statistical procedures.
3 experimental results.
Find through laboratory observation: negative control pipe test tube upper strata achromaticity and clarification, erythrocyte sink fully, not haemolysis; Positive pipe erythrocyte all dissolves, and solution colour is dark red, complete hemolysis.Solamargine injection 0.05mg/ml concentration pipe is haemolysis not, the clarification of 0.1mg/ml concentration test tube upper strata, but the erythrocyte sinking is not exclusively, and 0.15mg/ml concentration test tube erythrocyte partly sinks, and supernatant liquid takes on a red color, and certain haemolysis is arranged.Three concentration test tubes of solamargine liposome are haemolysis not all.The spectrophotography testing result is seen Fig. 2:
Can know by Fig. 2: the about 0.1mg/ml of solamargine injection haemolysis safe concentration; Two kinds of liposomees of solamargine still do not have haemolysis when concentration 0.15mg/ml, explain that Liposomal formulation can improve the haemolysis of solamargine injection to a certain extent.
Experiment 2.The blood vessel irritation experiment.
1 experiment material.
1.1 medicine.
The solamargine injection, concentration 0.76mg/ml; Solamargine liposome A (according to embodiment 1 preparation), 1.0mg/ml; Solamargine liposome B (according to embodiment 3 preparations), 1.0mg/ml.
1.2 animal.Livid purple blue rabbit
9Only, male and female are regardless of, body weight 1.8 ~ 2.2kg.
2 experimental techniques.
Get 9 of healthy livid purple blue rabbit, male and female are regardless of, and body weight 1.8～2.2kg is divided into 3 groups at random, and promptly solamargine injection group, solamargine liposome A group, solamargine liposome B organize 3 every group.
Give every day rabbit right auricular vein injection concentration be 0.1mg/ml solamargine injection or Liposomal formulation 5ml/ only, left auricular vein injection equivalent sodium chloride injection compares, continuous 3 days, before and after every ear administration with 75% alcohol disinfecting.2 rabbit are put to death in back 48 hours of last injection, cut administration bilateral ear, and 10% formalin is fixed, and makes the pathology sections observation.Remain 1 rabbit and continue to observe after 14 days, put to death, cut administration bilateral ear, 10% formalin is fixed, and makes the pathology sections observation.Angiological pathology inspection comprises blood vessel wall and endotheliocyte, thrombosis, inflammation, tissue variation, necrosis etc.
3 experimental results.
Dark red or the aubergine of solamargine injection group rabbit every auris dextra injection site blood vessel after the administration has significantly edema, does not see erosion; Administration finishes back about 12 days, and the injection site is replied normal; Liposome A and B group administration rabbit ear outward appearance and matched group relatively do not have significant change.
4 pathological examination results are following:
The administration phase.
Sodium chloride injection contrast ear: every rabbit ear drawn materials, and to locate the auricular vein tube wall complete, clear in structure.Blood vessel wall liner simple squamous epithelium, cell does not have swelling, or necrosis comes off.Do not see in the lumen of vessels that mural thrombus forms.Tube wall, Guan Zhouwu cell infiltration and bleeding.
Solamargine injection group: every rabbit ear drawn materials and located vascular endothelial cell degeneration, necrosis, comes off, and obvious thrombosis is arranged in the lumen of vessels, and Mild edema around blood vessel wall, the blood vessel has a small amount of cell infiltration.
Solamargine liposome A group: every rabbit ear drawn materials, and to locate the auricular vein tube wall complete, clear in structure.Blood vessel wall liner simple squamous epithelium, accidental coming off.Do not see in the lumen of vessels that mural thrombus forms.
Solamargine liposome B group: every rabbit ear drawn materials, and to locate the auricular vein tube wall complete, clear in structure.Blood vessel wall liner simple squamous epithelium, accidental coming off.Wall, Guan Zhouwu cell infiltration and bleeding.
Solamargine injection group: every rabbit ear drawn materials and located vascular endothelial cell degeneration, necrosis, comes off, no obvious thrombosis in the lumen of vessels, and blood vessel wall, blood vessel be Mild edema on every side, and a small amount of cell infiltration is arranged.
convalescent period.
Every rabbit ear drawn materials, and to locate the auricular vein tube wall complete, clear in structure.Blood vessel wall liner simple squamous epithelium cell does not have swelling, or necrosis comes off.Do not see in the lumen of vessels that mural thrombus forms.Guan Zhouwu cell infiltration and bleeding.
Can know that by The above results the effect of solamargine liposome vascular stimulation is weaker than normal injection.
Experiment 3.The muscle irritation experiment.
1 experiment material.
1.1 medicine.
The solamargine injection, concentration 0.76mg/ml; Solamargine liposome A (according to embodiment 1 preparation), 1.0mg/ml; Solamargine liposome B (according to embodiment 3 preparations), 1.0mg/ml.
1.2 animal.Livid purple blue rabbit
6Only, male being regardless of, body weight 1.8 ~ 2.2kg.
2 test methods.
Get 6 of healthy livid purple blue rabbit, body weight 1.8～2.2kg is divided into 3 groups at random, 2 every group.Sentence sterile working's method respectively at the right lateral thigh musculus quadriceps and inject three kinds of solamargine dosage form test sample 1ml, the opposite side same area is injected the isometric(al) sodium chloride injection and is compared.Every day 1 time, totally 3 times.Put to death rabbit behind the last administration 48h, dissect the back and take out quadriceps femoris, vertically cut, observe the IR of injection site, be converted into corresponding reaction score value by Fig. 3, and make the local organization pathological examination.
Calculate the summation of 6 quadriceps femoris order of reactions according to Fig. 3.If the highest and minimum difference of each quadriceps femoris order of reaction should be got 2 animals in addition and test again greater than 2 o'clock.
3 experimental results.
Every rabbit muscular irritation response situation is seen Fig. 4.Can know that by Fig. 46 rabbit muscular irritation reaction average response score values are below 2.
The pathological examination result is following:
The sodium chloride injection matched group: quadriceps femoris is a striped muscle, and the muscle fiber band is clear under the light microscopic, and a matter blood vessel does not have dilatation and congestion, and a quality is few, no acute, chronic inflammation cellular infiltration and bleeding.
Solamargine injection administration group: quadriceps femoris is not seen myofibrosis, necrosis, and a matter is not seen hyperemia, pathological changes such as hemorrhage, accidental cell infiltration disease.
Solamargine liposome group: quadriceps femoris is a striped muscle, and the muscle fiber band is clear under the light microscopic, and a matter blood vessel does not have dilatation and congestion, and a quality is few, no acute, chronic inflammation cellular infiltration and bleeding.
Can know that according to muscular irritation scoring and pathological examination the solamargine liposome is lighter than normal injection agent with muscular irritation under the concentration.
Experiment 4.Undue toxicity's experiment.
1 experiment material.
1.1 medicine.
The solamargine injection, concentration 0.76mg/ml; Solamargine liposome A (according to embodiment 1 preparation), 1.0mg/ml; Solamargine liposome B (according to embodiment 3 preparations), 1.0mg/ml.
1.2 animal.15 of ICR mices, ♀, 18～22g.
2 experimental techniques.
Get 15 of ICR mices, ♀, 18～22g; Be divided into three groups at random; Every group 5, only distinguish tail vein injection solamargine (0.1mg/kg), solamargine liposome A (0.1mg/kg), solamargine liposome B (0.1mg/kg) 0.5ml/, the reaction after the administration of observation mice.All mice must not have death in 48 hours, if any death, then should get 10 retrials of mice of body weight 18～19g in addition, and the retrial mice must not have death in 48 hours.
3 experimental results.
Three groups totally 15 mices in 24h and 48h, observe all normally, the no phenomena of mortality occur.Under this experiment condition, three kinds of preparation mice undue toxicity experiments are all qualified.
Experiment 5.The solamargine liposome is to rat liver cancer H22 transplanted tumor tumor-inhibiting action research experiment.
1 experiment material.
1.1 laboratory animal.
The solamargine injection, concentration 0.76mg/ml; Solamargine liposome A (according to embodiment 1 preparation), 1.0mg/ml; Solamargine liposome B (according to embodiment 3 preparations), 1.0mg/ml.
1.2 laboratory animal.50 of ICR mices, female, body weight 18-22g.
1.3 tumor line.Rat liver cancer H22, the tumor strain is gone down to posterity by mouse ascites.
2 experimental techniques.
The hepatocarcinoma H22 kind Mus that the ascites of learning from else's experience goes down to posterity takes off cervical vertebra and puts to death in the gnotobasis, milky ascites is extracted in abdominal part large tracts of land iodine tincture sterilization immediately, with normal saline according to 1: 4 diluted for use.Get 50 of ICR mices, female, respectively at the oncocyte suspension of its right side nape portion subcutaneous vaccination preparation, every inoculation 0.2ml, inoculation back 24h is divided into 5 groups to mice at random, that is: (1) model group (normal saline, iv); (2) the cyclophosphamide group (25mg/kg, ip); (3) the solamargine group (1.2mg/kg, iv); (4) solamargine liposome A group (1.2mg/kg, iv); (5) solamargine liposome B group (1.2mg/kg, iv).Every group 10.Divide into groups to begin administration the same day, once a day, administration is 8 times altogether, and mice is put to death in each administration 24 backs, peels off tumor tissues and weighs, and calculates administration group tumour inhibiting rate; Get mouse thymus and spleen simultaneously, calculate organ index comparable group differences; Just divide another name mice body weight in experiment, calculate mice weight increase rate with the experiment end.
3 experimental results.
3.1 the influence of and tumour inhibiting rate heavy to mouse tumor.
Can be known by Fig. 5: mouse tail vein injection gives solamargine 1.2mg/kg; The tumour inhibiting rate of injection dosage form is 51.16%; The two kinds of liposome A and the B tumour inhibiting rate of same dose are respectively 57.26% and 58.87%; Relatively tumor killing effect is better with former injection, possibly be because of liposome has certain targeting, can improve drug effect.
3.2 influence to the mice organ index.
Can be known by Fig. 6: thymus exponential sum index and spleen index all had certain reduction after mice gave the solamargine injection, and with model group relatively have significant difference (P 0.05, P 0.01), medicine demonstrates certain side effect under this dosage.And the solamargine liposome of same dose has only slight inhibition trend to mouse thymus and spleen, with model group there was no significant difference (P>0.05) relatively.It is littler that normal injection is compared in the side effect of results suggest solamargine liposome, more helps clinical use.
3.3 influence to the mice body weight.
Can be known by Fig. 7: can significantly suppress weight of mice behind the continuous tail vein injection of solamargine, with model group significant difference (P < 0.01) arranged relatively, the solamargine liposome of same dose does not then have obvious influence to the mice body weight.
Experiment 6.The solamargine liposome is studied ehrlich ascites tumor (EAC) transplanted tumor tumor-inhibiting action.
1 experiment material.
1.1 laboratory animal.
The solamargine injection, concentration 0.76mg/ml; Solamargine liposome A (according to embodiment 1 preparation), 1.0mg/ml; Solamargine liposome B (according to embodiment 3 preparations), 1.0mg/ml.
1.2 laboratory animal.50 of ICR mices, female, body weight 18-22g.
1.3 tumor line.The mice ehrlich ascites tumor, the tumor strain is gone down to posterity by mouse ascites.
2 experimental techniques.
The ascites of the learning from else's experience EAC kind Mus of going down to posterity takes off cervical vertebra and puts to death in the gnotobasis, milky ascites is extracted in abdominal part large tracts of land iodine tincture sterilization immediately, with normal saline according to 1 to 4 diluted for use.Get 50 of ICR mices, female, at the oncocyte suspension of its right side nape portion subcutaneous vaccination preparation, every inoculation 0.2ml inoculates back 24h and is divided into 5 groups to mice at random respectively, and promptly (1) model group (is given and normal saline, iv); (2) the cyclophosphamide group (is given and 25mg/kg, ip); (3) the solamargine group (1.2mg/kg, iv); (4) solamargine liposome A group (1.2mg/kg, iv); (5) solamargine liposome B group (1.2mg/kg, iv).Every group 10.Divide into groups to begin administration the same day, once a day, administration is 8 times altogether, and mice is put to death in each administration 24 backs, peels off tumor tissues and weighs, and calculates administration group tumour inhibiting rate; Get mouse thymus and spleen simultaneously, calculate organ index comparable group differences; Just divide another name mice body weight in experiment, calculate mice weight increase rate with the experiment end.
3 experimental results.
3.1 the influence of and tumour inhibiting rate heavy to mouse tumor.
Can be known by Fig. 8: mouse tail vein injection gives solamargine 1.2mg/kg; The tumour inhibiting rate of injection dosage form is 51.16%; The two kinds of liposome A and the B tumour inhibiting rate of same dose are respectively 62.25% and 62.35%, and relatively tumor killing effect is better with former injection, possibly be because of liposome certain targeting to be arranged; Can improve drug effect, the drug effect of two kinds of liposome prescriptions is suitable.
3.2 influence to the mice organ index.
Can be known by Fig. 9: thymus exponential sum index and spleen index all had certain reduction after mice gave the solamargine injection, but with model group there was no significant difference (P>0.05) was arranged relatively.The solamargine liposome of the identical administering mode of same dose has slight inhibition trend to mouse thymus, with model group there was no significant difference (P>0.05) relatively; Liposome does not have obvious influence to mouse spleen simultaneously.From data relatively, the solamargine liposome is weaker than the normal injection dosage form to the inhibitory action of mouse thymus and spleen, more helps clinical use.
3.3 influence to the mice body weight.
Can be known by Figure 10: the weight of mice rate is for can significantly suppress weight of mice behind the continuous tail vein injection of solamargine; With model group significant difference (P < 0.05) is arranged relatively, the solamargine liposome of same dose does not then have obvious influence to the mice body weight.
Experiment 7.The solamargine liposome is studied lewis lung cancer (Lewis) transplanted tumor tumor-inhibiting action.
1 experiment material.
1.1 laboratory animal.
The solamargine injection, concentration 0.76mg/ml; Solamargine liposome A (according to embodiment 1 preparation), 1.0mg/ml; Solamargine liposome B (according to embodiment 3 preparations), 1.0mg/ml.
1.2 laboratory animal.C
5750 of bl/6 mices, female, body weight 18-22g.
1.3 tumor line.The strain of mice lewis lung cancer (Lewis) tumor; In vitro culture.
2 experimental techniques.
Get the external lewis lung cancer cell that is in exponential phase, after the digestion washing, adjust cell concentration to 10 with the culture medium that does not contain serum
7Individual/ml.Get C
5750 of bl/6 mices, female, at the tumor cell suspension of its right side nape portion subcutaneous vaccination preparation, every inoculation 0.2ml inoculates back 24h and is divided into 5 groups to mice at random respectively, and promptly (1) model group (is given and normal saline, iv); (2) the cyclophosphamide group (is given and CTX25mg/kg, ip); (3) the solamargine group (1.2mg/kg, iv); (4) solamargine liposome A group (1.2mg/kg, iv); (5) solamargine liposome B group (1.2mg/kg, iv).Every group 10.Divide into groups to begin administration the same day, once a day, administration is 8 times altogether, and mice is put to death in each administration 24 backs, peels off tumor tissues and weighs, and calculates administration group tumour inhibiting rate; Get mouse thymus and spleen simultaneously, calculate organ index comparable group differences; Just divide another name mice body weight in experiment, calculate mice weight increase rate with the experiment end.
3 experimental results.
3.1 the influence of and tumour inhibiting rate heavy to mouse tumor.
Can be known by Figure 11: mouse tail vein injection gives solamargine 1.2mg/kg; The tumour inhibiting rate of injection dosage form is 47.69%; The two kinds of liposome A and the B tumour inhibiting rate of same dose are respectively 54.55% and 57.58%; Relatively tumor killing effect is better with former injection, possibly be because of liposome has certain targeting, can improve drug effect.
3.2 influence to the mice organ index.
Can be known by Figure 12: thymus exponential sum index and spleen index all had certain reduction after mice gave the solamargine injection, and medicine demonstrates the side effect of certain inhibition thymus and spleen under this dosage.And the solamargine liposome of the identical administering mode of same dose to the inhibitory action of mouse thymus and spleen less than normal injection.It is littler that normal injection is compared in the side effect of results suggest solamargine liposome.
3.3 influence to the mice body weight.
Can know by Figure 13: can significantly suppress weight of mice behind the continuous tail vein injection of solamargine; The body weight gain rate is 9.57%; With model group significant difference (P < 0.01) is arranged relatively, and the solamargine liposome group mice body weight of same dose and model group body weight gain rate are low slightly, but there was no significant difference (P 0.05); Prompting solamargine liposome is lower than normal injection agent to mice body weight inhibitory action, and side effect is littler.
Description of drawings
Fig. 1 is a hemolytic experiment application of sample scale.
Fig. 2 is spectrophotography haemolysis checking experiment result.
Fig. 3 is injection muscular irritation reaction standards of grading.
Fig. 4 is rabbit muscle irritation experiment muscular irritation response situation.
Fig. 5 is the influence of solamargine to tumor-bearing mice tumor weight and tumour inhibiting rate.
Fig. 6 is the influence of solamargine to the tumor-bearing mice organ index.
Fig. 7 is the influence of solamargine to the tumor-bearing mice body weight.
Fig. 8 is the influence of solamargine to tumor-bearing mice tumor weight and tumour inhibiting rate.
Fig. 9 is the influence of solamargine to the tumor-bearing mice organ index.
Figure 10 is the influence of solamargine to the tumor-bearing mice body weight.
Figure 11 is the influence of solamargine to tumor-bearing mice tumor weight and tumour inhibiting rate.
Figure 12 is the influence of solamargine to the tumor-bearing mice organ index.
Figure 13 is the influence of solamargine to the tumor-bearing mice body weight.
The specific embodiment
Below further describe concrete technical scheme of the present invention,, and do not constitute restriction its right so that those skilled in the art understands the present invention further.
Embodiment 1.The solamargine liposome preparation.
Prescription:
Solamargine 0.1g
Soybean lecithin for injection 1.5g
Cholesterol 0.4g
Tween 80 0.2g
PH=4.9 phosphate buffered saline 100ml
The soybean lecithin for injection, the cholesterol that take by weighing recipe quantity are dissolved in the 300ml ether, and solamargine is dissolved in the 100ml phosphate buffered saline, organic facies and water mix homogeneously; The tween 80 that adds recipe quantity again, heating in water bath to 35 ℃, supersound process 15min; Reduction vaporization falls organic solvent to there not being the ether flavor; Ultrasonicly again make its dispersion, promptly get the solamargine liposome, envelop rate 61.5%.
Embodiment 2.The solamargine liposome preparation.
Prescription:
Solamargine 0.1g
Soybean lecithin for injection 1.5g
Cholesterol 0.4g
Tween 80 0.2g
PH=4.9 phosphate buffered saline 100ml
The soybean lecithin for injection, the cholesterol that take by weighing recipe quantity are dissolved in the 300ml chloroform, are transferred in the round-bottomed flask, under 40 ℃ bath temperature; Rotating speed is 150r/min, and reduction vaporization falls the organic solvent film forming, treat that film forms after; Inflated with nitrogen number minute is removed organic solvent fully.Take by weighing recipe quantity solamargine and tween 80 and be dissolved in the 100ml phosphate buffered saline, buffer is changed in the flask, room temperature, normal pressure rotation are down washed film; Aquation 30min, supersound process 20min again come off fully to thin film; Promptly get the solamargine liposome, envelop rate 59.3%.
Embodiment 3.The solamargine liposome preparation.
Prescription:
Solamargine 0.1g
Soybean lecithin for injection 1.5g
Cholesterol 0.4g
Poloxamer F-68 0.2g
PH=4.9 phosphate buffered saline 100ml
The soybean lecithin for injection, the cholesterol that take by weighing recipe quantity are dissolved in the 300ml ether, and poloxamer F-68, solamargine are dissolved in the 100ml phosphate buffered saline, organic facies and water mix homogeneously; Heating in water bath to 35 ℃; Supersound process 15 minutes, reduction vaporization are fallen organic solvent to there being ether flavor, ultrasonicly again make its dispersion; Promptly get the solamargine liposome, envelop rate 70.5%.
Embodiment 4.The solamargine liposome preparation.
Prescription:
Solamargine 0.1g
Soybean lecithin for injection 1.5g
Cholesterol 0.4g
Tween 80 0.2g
PH=5.0 citrate buffer salt solution 100ml
Soybean lecithin for injection, the cholesterol of recipe quantity are dissolved in the 20ml ether; Slowly drop in the 100ml citrate buffer salt solution that is dissolved with solamargine and tween 80, rate of addition 1ml/min dropwises continued and stirs 2h; Eliminate organic solvent; Remaining liq supersound process 15min promptly gets the solamargine liposome, envelop rate 62.9%
Embodiment 5.The preparation of solamargine lipidosome injection.
Each described solamargine liposome among the embodiment 1-4 is crossed 0.22 μ m microporous filter membrane, embedding, inflated with nitrogen, sterilization gets the solamargine lipidosome injection.
Embodiment 6.The preparation of solamargine liposome powder for injection.
Each described solamargine liposome among the embodiment 1-4 is added 10% mannitol, cross 0.22 μ m microporous filter membrane, packing, lyophilization gets injectable powder.
Embodiment 7.A kind of solamargine liposome, it is made up of the following weight proportion raw material,
Described solamargine is commercially available solamargine extract, and wherein the solamargine content of monomer is 95%; Described phospholipid is Ovum Gallus domesticus Flavus lecithin; Described emulsifying agent is a poloxamer.
Described solamargine liposome can adopt the method for preparing of conventional liposome to process.
Embodiment 8.A kind of solamargine liposome, it is made up of the following weight proportion raw material,
Cholesterol 20; Emulsifying agent 10.
Described solamargine is commercially available solamargine extract, and wherein the solamargine content of monomer is 90%; Described phospholipid is hydrogenated soy phosphatidyl choline; Described emulsifying agent is a Polyethylene Glycol;
The concrete steps of its preparation method are:
(1) use water for injection secure ph is 3 faintly acid buffer salt solution, and solamargine, emulsifying agent are dissolved in this buffer, gets water;
(2) take by weighing phospholipid, cholesterol in said ratio, be dissolved in the organic solvent, get organic facies;
(3) with water with after organic facies is mixed, adopt reverse evaporation to handle earlier, ultrasonicly then make its dispersion, promptly get the solamargine liposome.
Embodiment 9.A kind of solamargine liposome, it is made up of the following weight proportion raw material,
Described solamargine is commercially available solamargine extract, and wherein the solamargine content of monomer is 98%; Described phospholipid is hydrogenated yolk lecithin; Described emulsifying agent is N, dinethylformamide.
The concrete steps of its preparation method are:
(1) use water for injection secure ph is 6.5 faintly acid buffer salt solution, and solamargine, emulsifying agent are dissolved in this buffer, gets water;
(2) take by weighing phospholipid, cholesterol in said ratio, be dissolved in the organic solvent, get organic facies;
(3) with water with after organic facies is mixed, adopt film dispersion method to handle earlier, ultrasonicly then make its dispersion, promptly get the solamargine liposome.
Embodiment 10.A kind of solamargine liposome, it is made up of the following weight proportion raw material,
Described solamargine is commercially available solamargine extract, and wherein the solamargine content of monomer is 80%; The two Semen Myristicae phosphatidylcholines of described phospholipid; Described emulsifying agent is the mixture of tween and poloxamer.
The concrete steps of its preparation method are:
(1) use water for injection secure ph is 4 faintly acid buffer salt solution, and solamargine, emulsifying agent are dissolved in this buffer, gets water;
(2) take by weighing phospholipid, cholesterol in said ratio, be dissolved in the organic solvent, get organic facies;
(3) with water with after organic facies is mixed, adopt injection method to handle earlier, ultrasonicly then make its dispersion, promptly get the solamargine liposome.
Embodiment 11.A kind of solamargine liposome, it is made up of the following weight proportion raw material,
Cholesterol 5; Emulsifying agent 5.
Described solamargine is commercially available solamargine extract, and wherein the solamargine content of monomer is 85%; Described phospholipid is two Semen Myristicae phosphatidyl glycerols; The mixture of described emulsifier tween and Polyethylene Glycol.
The concrete steps of its preparation method are:
(1) use water for injection secure ph is 5 faintly acid buffer salt solution, and solamargine, emulsifying agent are dissolved in this buffer, gets water;
(2) take by weighing phospholipid, cholesterol in said ratio, be dissolved in the organic solvent, get organic facies;
(3) with water with after organic facies is mixed, adopt reverse evaporation or film dispersion method or injection method to handle earlier, ultrasonicly then make its dispersion, promptly get the solamargine liposome.
Embodiment 12.A kind of solamargine liposome, it is made up of the following weight proportion raw material,
Cholesterol 7; Emulsifying agent 4.
Described solamargine is the solamargine monomer; Described phospholipid is two Semen Myristicae phosphatidylcholines or two Semen Myristicae phosphatidyl glycerol; Described emulsifying agent is tween and N, the mixture of dinethylformamide.
The concrete steps of its preparation method are:
(1) use water for injection secure ph is 3 faintly acid buffer salt solution, and emulsifying agent is dissolved in this buffer, gets water;
(2) take by weighing phospholipid, cholesterol, solamargine in said ratio, be dissolved in the organic solvent, get organic facies;
(3) with water with after organic facies is mixed, adopt reverse evaporation or film dispersion method or injection method to handle earlier, ultrasonicly then make its dispersion, promptly get the solamargine liposome.
Embodiment 13.A kind of solamargine liposome, it is made up of the following weight proportion raw material,
Described solamargine is the solamargine monomer; Described phospholipid is dioleoyl phospholipid phatidylcholine, or two palmityl phosphatidic acid; Described emulsifying agent is the mixture of poloxamer and Polyethylene Glycol.
The concrete steps of its preparation method are:
(1) use water for injection secure ph is 6.5 faintly acid buffer salt solution, and emulsifying agent is dissolved in this buffer, gets water;
(2) take by weighing phospholipid, cholesterol, solamargine in said ratio, be dissolved in the organic solvent, get organic facies;
(3) with water with after organic facies is mixed, adopt reverse evaporation or film dispersion method or injection method to handle earlier, ultrasonicly then make its dispersion, promptly get the solamargine liposome.
Described faintly acid buffer salt solution is PBS or citrate buffer solution; Described organic solvent is a kind of in chloroform, ether, dichloromethane, normal hexane, ethanol, the methanol.
Embodiment 14.A kind of solamargine liposome, it is made up of the following weight proportion raw material,
Cholesterol 15; Emulsifying agent 3.
Described solamargine is commercially available solamargine extract, and wherein the solamargine content of monomer is 87%; Described phospholipid is two palmityl phosphatidyl cholines; Described emulsifying agent is poloxamer and N, the mixture of dinethylformamide.
The concrete steps of its preparation method are:
(1) use water for injection secure ph is 4 faintly acid buffer salt solution, and emulsifying agent is dissolved in this buffer, gets water;
(2) take by weighing phospholipid, cholesterol, solamargine in said ratio, be dissolved in the organic solvent, get organic facies;
(3) with water with after organic facies is mixed, adopt reverse evaporation or film dispersion method or injection method to handle earlier, ultrasonicly then make its dispersion, promptly get the solamargine liposome.
Described faintly acid buffer salt solution is sodium hydrogen phosphate-potassium phosphate buffer.Described organic solvent is the mixed liquor of any two kinds of compositions in chloroform, ether, dichloromethane, normal hexane, ethanol, the methanol.
Embodiment 15.A kind of solamargine liposome, it is made up of the following weight proportion raw material,
Described solamargine is commercially available solamargine extract, and wherein the solamargine content of monomer is 96%; Described phospholipid is two palmityl phosphatidyl glycerols; Described emulsifying agent Polyethylene Glycol and N, the mixture of dinethylformamide.
The concrete steps of its preparation method are:
(1) use water for injection secure ph is 4.5 faintly acid buffer salt solution, and emulsifying agent is dissolved in this buffer, gets water;
(2) take by weighing phospholipid, cholesterol, solamargine in said ratio, be dissolved in the organic solvent, get organic facies;
(3) with water with after organic facies is mixed, adopt reverse evaporation or film dispersion method or injection method to handle earlier, ultrasonicly then make its dispersion, promptly get the solamargine liposome.
Described faintly acid buffer salt solution is sodium hydrogen phosphate-citrate buffer solution; Described organic solvent is the mixed liquor of any three kinds of compositions in chloroform, ether, dichloromethane, normal hexane, ethanol, the methanol.
Embodiment 16.A kind of solamargine liposome, it is made up of the following weight proportion raw material,
Cholesterol 14; Emulsifying agent 5.
Described solamargine is commercially available solamargine extract, and wherein the solamargine content of monomer is 92%; Described phospholipid is natural extract phospholipid, semi-synthetic or complete synthesis phospholipid; Described emulsifying agent is selected from tween, poloxamer, Polyethylene Glycol or N, one or more in the dinethylformamide.
The concrete steps of its preparation method are:
(1) use water for injection secure ph is 5.5 faintly acid buffer salt solution, and emulsifying agent is dissolved in this buffer, gets water;
(2) take by weighing phospholipid, cholesterol, solamargine in said ratio, be dissolved in the organic solvent, get organic facies;
(3) with water with after organic facies is mixed, adopt reverse evaporation or film dispersion method or injection method to handle earlier, ultrasonicly then make its dispersion, promptly get the solamargine liposome.
Described faintly acid buffer salt solution is the citric acid-sodium citrate buffer; Described organic solvent is selected from chloroform, ether, dichloromethane, normal hexane, ethanol or methanol.
Claims (10)
1. solamargine liposome, it is characterized in that: it is made up of the following weight proportion raw material,
Solamargine 1; Phospholipid 8～80;
Cholesterol 2～20; Emulsifying agent 1～10.
2. solamargine liposome according to claim 1 is characterized in that: the weight proportion of each raw material is:
Solamargine 1; Phosphatidase 11 5～25;
Cholesterol 2～10; Emulsifying agent 2～8.
3. solamargine liposome according to claim 1 is characterized in that: described phospholipid is natural extract phospholipid, semi-synthetic or complete synthesis phospholipid.
4. solamargine liposome according to claim 3 is characterized in that: described phospholipid is selected from: soybean lecithin, Ovum Gallus domesticus Flavus lecithin, hydrogenated soy phosphatidyl choline, hydrogenated yolk lecithin, two Semen Myristicae phosphatidylcholine, two Semen Myristicae phosphatidyl glycerol, dioleoyl phospholipid phatidylcholine, two palmityl phosphatidic acid, two palmityl phosphatidyl cholines or two palmityl phosphatidyl glycerols.
5. solamargine liposome according to claim 1 is characterized in that: described emulsifying agent is selected from tween, poloxamer, Polyethylene Glycol or N, one or more in the dinethylformamide.
6. the method for preparing like any one described solamargine liposome of claim 1-5 is characterized in that, its concrete steps are:
(1) use water for injection secure ph is 3～6.5 faintly acid buffer salt solution, and solamargine, emulsifying agent are dissolved in this buffer, gets water;
(2) take by weighing phospholipid, cholesterol in said ratio, be dissolved in the organic solvent, get organic facies;
(3) with water with after organic facies is mixed, adopt reverse evaporation or film dispersion method or injection method to handle earlier, ultrasonicly then make its dispersion, promptly get the solamargine liposome.
7. the method for preparing like any one described solamargine liposome of claim 1-5 is characterized in that, its concrete steps are:
(1) use water for injection secure ph is 3～6.5 faintly acid buffer salt solution, and emulsifying agent is dissolved in this buffer, gets water;
(2) take by weighing phospholipid, cholesterol, solamargine in said ratio, be dissolved in the organic solvent, get organic facies;
(3) with water with after organic facies is mixed, adopt reverse evaporation or film dispersion method or injection method to handle earlier, ultrasonicly then make its dispersion, promptly get the solamargine liposome.
8. according to the method for preparing of claim 6 or 7 described solamargine liposomees, it is characterized in that: described faintly acid buffer salt solution is PBS, citrate buffer solution.
9. according to claim 6 or 7 described solamargine liposomees, it is characterized in that: described organic solvent is selected from the mixed liquor of one or more compositions in chloroform, ether, dichloromethane, normal hexane, ethanol, the methanol.
10. the application of any one described solamargine liposome in preparation treatment hepatocarcinoma, lung cancer drugs in the claim 1～5.
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